Horseradish peroxidase labeled anti rabbit igg secondary antibody

Western blotting was performed as described previously (17 (link),18 (link)). Briefly, total proteins were extracted from fresh frozen tissues by RIPA lysis buffer (product code P0013B; Beyotime Institute of Biotechnology). Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Protein samples (30 µg) were then separated electrophoretically using 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels and transferred to nitrocellulose membranes (EMD Millipore). After blocking the nonspecific binding sites with 5% nonfat milk diluted in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature, the membranes were blotted with a rabbit polyclonal anti-RBMS3 antibody (1:2,000; product code ab198248; Abcam) at 4°C overnight. After three 10-min TBST washes, the membranes were blotted with horseradish peroxidase-labeled anti-rabbit IgG secondary antibody (product no. 7074P2; Cell Signaling Technology) at a dilution of 1:3,000 at room temperature for 60 min. The membranes were then washed three times with TBST for 10 min each time, and the bound antibodies were developed using the enhanced chemiluminescence system (product code 34577; Thermo Fisher Scientific, Inc.). GAPDH was detected as a loading control using an anti-GAPDH antibody (1:3,000; Ab103-02; Vazyme).